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Editorial
. 2010 Dec;95(12):1985-8.
doi: 10.3324/haematol.2010.033225.

Erythroblast enucleation

Anna Rita Migliaccio
Editorial

Erythroblast enucleation

Anna Rita Migliaccio. Haematologica. 2010 Dec.
No abstract available

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Figures

Figure 1.
Figure 1.
Diagrammatic scheme of the interaction between an erythrocyte and a macrophage during the process of enucleation. The alterations in the structural proteins of the plasma membrane and of the cytoplasmic filaments occurring during erythroid maturation disrupt the connection between the nucleus, the plasma membrane and the centrosome. The centrosome, therefore, becomes unable to drive dissolution of the nuclear membrane, to organize the fiber-chromosome spindle or to apply the tension necessary to divide the cell into two distinct elements. It is speculated that the tension necessary to divide the erythrocyte into a reticulocyte and a pyrenocyte is applied by the macrophage (or the fibronectin mesh) through cell-cell interactions with the pole of the erythrocyte membrane containing the receptors required for macrophage interaction. The insert shows an erythroid island formed by human erythroid cells and a macrophage at day 10 of ex-vivo culture (magnification 40X). The background is an electroimmuno-gold staining with fibronectin-specific antibody (the dots) of marrow mesh from a mouse (magnification 20,000X). The adhesion molecules mediating the interactions between erythroid cells and the macrophage are drawn from Chasis et al. The diagram of the cytoskeleton elements connecting the centrosome (yellow circle) with the nuclear membrane and with the chromosome centromere are from Razafsky et al. These connections are interrupted in erythrocytes.
Figure 2.
Figure 2.
HDAC isoforms (A) and HDAC complexes (B) involved in erythroid maturation. The earliest recognizable erythroid cell, the proery-throblast, is capable of self-replication and of maturation into basophilic erythroblasts. Once nuclear condensation is completed, the orthrochromatic erythroblast undergoes enucleation, a process that generates two daughter structures, the reticulocyte, which contains most of the cytoplasm, and the pyrenocyte, which contains the nucleus surrounded by a small cytoplasmic ring. HDAC1, HDAC3 and HDAC2 have been identified to regulate the decision between self-replication and maturation, the switch from γ- to β-globin expression and chromatin condensation in preparation for enucleation, respectively. (i) Cell proliferation: two GATA transcription factors, GATA2 and GATA1, control proliferation and maturation of erythroid cells. When expression of GATA2 is greater than that of GATA1, erythroid cells proliferate while when GATA1 expression becomes predominant, cells mature. GATA2 and GATA1 regulate each other’s expression. GATA2 activates GATA1 expression while GATA1, once expressed, up-regulates its own expression and suppresses that of GATA2. GATA1 suppresses GATA2 expression by docking to the regulatory region of the gene a complex containing HDAC1. By deacetylating the histones, the complex closes the chromatin configuration of the locus which is no longer recognized and transcribed by the polymerase complex. Biochemical studies coupled with loss of function studies in the mice have identified that GATA1 binds the complex indirectly through its obligatory partner FOG1 which contains a binding domain for HDAC1. The insert in (B) shows an immuno-precipitation with a GATA1-specific antibody of protein extracts from ex-vivo expanded immature (iEBs) and mature (mEBs) human erythroblasts analyzed by western blot for the presence of GATA1 and HDAC1. These data confirm that GATA1 and HDAC1 are also associated in mature human erythroblasts, suggesting that this complex may suppress GATA2 expression (and proliferation) also in these cells. (ii) Hemoglobin switching: the observation of specific histone acetylation patterns during globin switching in mice has suggested that HDAC may participate in the silencing complex that represses γ-globin gene expression during erythroid maturation.v Proof-of-concept for HDAC involvement in repression of γ-globin expression was further provided by the observation that the HDAC inhibitor (HDACi) butyrate delays the HbF to HbA switch in sheep fetuses and induces HbF synthesis in human erythroid cultures, in adult baboons, in some patients with β-thalassemia and in most patients with sickle cell disease. siRNA-mediated loss of function experiments have recently indicated that the isoform which specifically suppresses HbF synthesis in human erythroid cells and that is targeted by butyrate is HDAC3. More recent genetic and mass spectrometry studies have identified that the specificity of the silencing may be provided by recruitment to the complex of BCL11A which docks the HDAC to the γ-globin regulatory region. (iii) Chromatin condensation: in this issue of the journal Ji et al. describe that chromatin condensation in preparation for orthochromatic erythroblast formation is regulated by HDAC2. It is possible that, by deacetylating the centromere-specific histone H3.3, HDAC2 is also responsible for dissociation of the centromeres from the fibers of the spindle. The docking protein for HDAC2 to the centromere has not been identified yet.
Figure 3.
Figure 3.
Pharmacophore model of HDAC inhibitors (HDACi) and the chemical structures of a FDA-approved HDACi (SAHA) and of two representative new generation HDACi. The effects of these two compounds on enucleation of human erythroblasts cultured in the presence of erythropoietin are presented on the left. A compound inhibits HDAC activity by irreversibly binding to the catalytic domain of the enzyme. To bind to the catalytic domain, its chemical structure should resemble either the substrate (the acetylated lysine of the proteins) or the product (the acetate ion) of the enzymatic reaction. The pharmacophore model for HDACi, such as SAHA or trichostatin A, which mimic the structure of the substrate includes four domains: a zinc binding group (ZBG), a hydrophobic spacer (HS), a connection unit (CU) and an interaction domain with the rim of the catalytic pocket of the enzyme (CAP)., By altering the chemical residues of these domains, pharmaceutical chemists are synthesizing new generation HDACi, such as the compound aroyl-pyrrolyl hydroxyl-amide 9 (APHA 9) and uracyl-based hydroxyl-amide 24 (UBHA 24) described in this Figure. The inhibitory activity (ID50) of SAHA, APHA 9 and UBHA 24 against purified human HDAC4 and HDAC1, used as examples of class II and class I HDAC, is reported, for comparison. APHA 9 and UBHA 24 were identified by screening a library of 24 new HDACi for their ability to reactivate γ-globin expression in erythroblasts generated ex-vivo from normal donors and from β°-thalassemic patients. The paper by Ji et al. led us to perform re-analyses for signs of enucleation in May-Grunwald-Giemsa stained smears prepared from cells obtained in the course of this previous study. Pyrenocytes (arrows) and ghosts of reticulocytes (arrow-head, reticulocytes do not survive the shear force of the centrifugation process) were easily detectable on smears of human erythroblasts induced to mature with erythropoietin for 4 days (control). Nuclear condensation and enucleation were, instead, greatly inhibited by addition of APHA 9, suggesting that HDAC are also required for chromatin condensation of human erythroblasts. However, the presence of UBHA 24 had no apparent effect on enucleation of human erythroblasts in culture. These results indicate that it should be possible to identify therapeutically active HDACi which may not induce anemia because they do not target HDAC2 and do not suppress enucleation. Magnification: 40X.
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References

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